Method and apparatus for culturing bacteria on solidified culture media



Qct. 26, 1937. R THOMPSON 2,096,866 METHOD AND APPARATUS FOR CUL'IfURINGBACTERIA ON SOLIDIFIED CULTURE MEDIA Filed March 22, 1934 2'Sheets-Sheet l FIGJ INVENTOPL ROBERT-R-THOMPSON I fizfwmw ATTORNEY Oct.26, 1937, R. R. THOMPSON 2,096,866

METHbD AND APPARATUS FOR CULTURING BACTERIA ON SOLIDIFIED CULTURE MEDIAFiled March 22, 1934' 2 Sheets- Sheetfi Fl LM INVENTOR-ROBER'IR-THOMPSON ATTORNEY FIG. 2

Patented Oct. 26, 1937 2,096,866 METHOD! AND APPARATUS 'FoR' CULTUR- INGBACTERIA ON MEDIA SOLIDIFIED CULTURE Robert Redvers Thompson,Ganan'oque, Ontario,

' Canada i Application March 22,

4 Claims.

This invention relates to an improved lmethod and apparatus for use inconnection, with; .the

culturing of bacteria on the surface of solidified culture media.

When culturing certain organisms it is desirable to inoculate onto thesurface of solidified culture medium. The present practice of usingPetri plates for this work is quite satisfactory when the culturing iscarried on in a well equipped laboratory and when it is possible todeliver the inoculum to the laboratory in a fresh condition. Casesarise, however, where these requirements cannot be met and where the useof Petri plates gives rise to Various difficulties. For example, whenbacteriological investigations are carried out at locations hundreds ofmiles distant from the base laboratory, it is frequently impossible totransport inoculum, such as blood or milk, to the laboratory in thecondition necessary to ensure a satisfactory bacteriological analysis.In such cases poured Petri plates have been used for carrying out thebacteriological investigation at the place of infection but it was founddifficult to prevent contamination of the poured plates duringtransportation and to obtain satisfactory inoculation and distributionof inoculum in available places such as private homes and poorlyequipped laboratories. Contamination, subsequent to inoculation, isalso'a 0 troublesome factor in connection with the use of Petri plates,since these plates have'to beincubated for periods of two to three weekswhen culturing the organisms from blood.

The present invention overcomes the above mentioned difficulties by theprovision of a method and apparatus which makes feasible the employmentof glass test-tubes (commonly called rolled-tubes), in place of Petrivplates, for surface inoculation of solidified culture media. In additionto lessening the risk of contamination, during transportation and use,rolled tubes are more convenient to carry than Petri plates and are muchcheaper.

The roll-tube method of culturing bacteria, as heretofore practiced,consists in adding the inoculum to liquefied culture medium contained inthe tube and then rotating the tube-to spread the inoculated culturemedium in the form of a film. The tubes are usually rotated by handwhile lying in a horizontal plane. More recently, there has beendeveloped a mechanical tube spinning machine which spreads theinoculated culture medium over the walls of the tube by centrifugalforce. of turning out uniform culture tubes free from This machine hasthe advantage 1e34, Serial No. 716,922

wrinkles and is more rapid than the hand rolling, However, so faras-I amaware, none of the previous workers in this field have developed anymethod or apparatus which makes feasible theemplo-yment of rolled tubes,instead of Petri plates, for culturing bacteria on the surface of apreviously prepared film of culture medium.

According to the present invention, a tube, containing the culturemedium in liquid form, is supported in an upright position in a body ofcooling liquid with the lower end of the tube received in the cavity ofa rotary tube spinning cup. When the cup is rotated at a sufficientlyhigh rate ofspeed (usually above 1500 R. P. M.) the centrifugal forcedeveloped causes the culture medium to spread over the walls of thespinning .tube to form a solidified film gradually diminishing inthickness toward the upper limit.

The tube is then removed and stored away ina cold place until theinoculum is to be added and distributed over the surface of the preparedfilm. It is desirable that the spreading of the inoculum be ccnducivetothe growing of well isolatedcol- :onies at some location on the preparedfilm. This is, accomplished in the following manner. After addition ofthe inoculum the prepared tube is dropped into the cooling liquid andengaged with the spinning cup which is made to revolve at a speed ofabout 2640 R. P. M. Owing to the centrifugal force developed at thisspeed and to the sloping surface of the film the inoculum is causedtoclimb up the film to a point near its upper limit. With'this procedurethe density of the seeding diminishes toward the upper limit of the filmand ensures the growing of well isolated colonies.

Another feature of this invention resides in a peculiar hydraulicclutching action resulting from the submergence of the tube spinning cupin a casing containing the cooling liquid into which the tube is droppedthrough a suitable opening or tube guide aligned with the cup. With thisassembly the tube floats in the liquid out of contact with the cup whenthe latter is at rest or rotating at a slow speed but, in the case ofthe particular apparatus illustrated, is automaticallydrawn down intothe cupby the action of the disturbance setup in the cooling liquid whenthe cup is rotated at relatively high rates of speed ranging upwardsfrom about 1500 R. P. M. When the cup is slowed down or brought to restthe actionof the liquid is such that the tube is then floated out of thecup toa released position re- .mote therefrom. It will thus be seen thatthe cupand tube constitute driving and driven members which are clutchedtogether or released by the action of the cooling liquid in response tovariations in the operating speed of the cup. Obviously this feature ofthe present invention is susceptible of wide application in themechanical arts since the tube may be replaced by another. form ofdriven member adapted to actuate some mechanical ccntrivance when movedto Land from the driving cup by the action of the Figure 3 is a View inlongitudinal section of a rolled tube showing the manner in which theculture medium is distributed in accordance with this invention to forma film of varying thickness.

Referring more particularly to the drawings, 5 designates a casingcomprising a cast metal base 6, a cover member '7 and a glass cylinder8, the latter being clamped between the base and cover by a series ofclamping bolts 9. The base 6 is provided with a shaft bearing l0 and awater inlet passage I] and is slidably mounted on a supporting standard[2 by means of the arm [3, sleeve M and clamping screw I5. The covermember 7 is provided with a neck extension I6 equipped with an overflowconnection I1.

The casing 5 encloses a driving cup l8 provided with a depending shaftI9 extending downwardly through the bearing l0.' A pulley 20 at thelower end of this shaft is driven, by a belt 2|, from a pulley 22 on thearmature shaft of an electric motor 23. As shown more particularly inFigure 2 the cup I8 is slightly conical and is provided with a frictionlining 24 of rubber or other suitable material. The bottom of this cuprests in a recess 25 at the upper end of the shaft bearing It.

A cylindrical tube guide 26 is fitted in the neck extension I6 of thecover member in axial alignment with the cup l8. This tube guide isprovided with a liner or bushing 26a and is adjustably mounted on thestandard I! by means of the arm 21, sleeve 28 and clamping screw 29. Thelower end of the standard I2 is screwed into a fiat base 30 mounted on amain supporting plate 3| which also carries the motor 23.

A constant flow of water through the casing 5 is maintained by means ofa Water supply pipe 32 and an overflow pipe 33, the former beingconnected with the water passage H of the base 6 and the latter with theoverflow connection I! of the cover member. is controlled by a valve 34mounted on the main supporting plate 31 and adapted for connection withany suitable water supply.

The manner in which the above mentioned apparatus is used in preparingrolled tubes for surface inoculation will now be described. A rolledtube 35, containing the culture medium in liquid form, is droppedthrough the tube guide 26 into the body of water contained in the casing5. If the cup I8 is at rest or turning at a relatively slow speed, thetube '35 simply floats in the liquid in the vertical position indicatedby dotted lines in Figure 2. If, now, the rotational speed of the cup issufficiently accelerated The flow through pipe 32 This shaping of thefilm is clearly shown in Figure 3 and has certain advantages inconnection with the subsequent distribution of inoculum as hereinafterexplained. The time required to form the film is usually about one andone-half minutes after which the tube may be removed and placed in acold room until it is to be used for culturing. It will thus be seenthat a large number of tubes may be quickly prepared in accordancewiththis invention and stored away for future-use with very little danger ofcontami-.

nation provided reasonable care is exercised by the operator) The cup I8is preferably slowed down or brought to rest before removing theprepared tube. When the turning speed of the cup drops below a certainvalue, the tube is automatically floated out of driving engagement withthe cup and resumes the dotted line position shown in Figure 2. Theoperator then grasps the projecting part of the tube and removes it fromthe casing 5.

From the foregoing it will be seen that this invention presents, as oneof its characteristic features, a tube spinning apparatus in which theliquid employed for cooling the rolled tube durreleased when the speedof the driving member or cup falls below that required for the spinningoperation. It is also desired to point out that, in addition to its usein connection with the present invention, the aforesaid hydraulic clutchaction is obviously susceptible of wide application in themechanicalarts since the rolled. tube 35 may obviously be replaced byanother suitable form of driven member intended to actuate somemechanical device when moved into and out of the driving cup in responseto the action of the liquid disturbance caused by rotation of the cup atdifferent speeds. the liquid disturbance upon which the hydraulicclutching action in question is based is not en- While the exact natureof" tirely free from uncertainty, it is believed that the following is acorrect description of what actually takes place.

7 When rotated at a relatively high rate of speed,

the driving cup apparently sets up a disturbance which causes thecooling liquid to flow out of the cup at its edges or periphery and inat the center, thus lowering the hydraulic pressure along the the axisof the cup apparently unbalances the forces holding the rolled tube insuspension so that the tube is automatically forced down into the cupand caused to rotate, by frictional contact, with the rubber lining.With a decrease in speed the liquid circulation in the cup is ap-'parently reversed since the test tube is automatically floated out ofthe cup when the speed of the latter falls below a certain value. By'

axis of the cup. This decrease in pressure along actual tests it hasbeen ascertained that, at a speed of 1500 revolutions, there is adecrease in pressure ,of 2.54 centimeters of water at a pointapproximately one-half inch above the center of the driving cup. .Thiswould be equivalent to the rolled tube floating 2.54 centimeters lowerin the water which, in the case of the construction illustrated in thedrawings, is sufficient to bring the tube down into the cup. At higherspeeds the decrease in pressure along the central axis of the cup isgreater and would, of course, exert a greater downward pull on therolled tube. I have also found that the hydraulic clutching effect duetothe liquid circulation set up within the driving cup 18 may beenhanced by providing the cup with flow openings near its bottom wall,as indicated at 31' in Figure 2. When these flow openings are providedthe downward pull on the rolled tube is stronger for a given speed ofcup rotation than when they are omitted. 7

We come now to the mode of using the described apparatus in connectionwith the distribution of inoculum over the surface of the filmscontained in the previously prepared tubes. The inoculum is preferablyadded to a prepared tube by means of a pipette or hypodermic needle. If

desired the inoculum may be thus added to a whole series of preparedtubes before resorting to the use of the spinning apparatus fordistributing the inoculum over the prepared films. In the spreading ofthe inoculum it is desirable to have uniformity and to effect thespreading in a manner that will be conducive to the growing of wellisolated colonies which permit of easy picking at some location on thefilm.. These results are obtained by dropping the prepared tube,containing the inoculum, into the body of water in the casing 5 when thedriving cup I8 is rotating at a speed of about 2640 R. P. M. Thecentrifugal force developed at this speed forces the inoculum to climbup the sloping exposed surface of the cone-shaped film to a point nearthe upper limit of the film. As the inoculum is forced upward the filmis seeded with the organisms therefrom. This procedure results in themost dense seeding at the base of the film and a gradual lessening ofthe density of the seeding towards the upper limit of the film. Theadvantage of' this is that well isolated colonies are obtained towardsthe upper limits of the film. After the inoculum has been thusdistributed the tube is removed and placed in an incubator where it isheld at the desired temperature.

Having thus described my invention, what I claim is:

1. Apparatus of the character described comprising a stationary casinghaving an opening at the top for the insertion of a test-tube in anupright position, a cup shaped member rotatably mounted in the casing inline with said opening to receive the lower end of the test-tube, tubeencircling means located above the opening in the casing adapted toassist in maintaining the upright position of the tube withoutinterfering with rotation thereof, means for maintaining said casingfilled with liquid to a level substantially above the top of the cupshaped member and means for rotating said cup.

2. Bacteriological apparatus comprising an upright stationary casinghaving an opening at the top to permit a test-tube to be inserted in thecasing in an upright position, a test-tube supporting member in linewith said openin adapted to encircle and support the upper portions ofthe test-tube without interfering with rotation thereof, a cup shapedmember rotatably mounted in the lower portion of the casing in line withsaid opening and adapted to receive the lower end of the test-tubetherein, means for maintaining said casing filled with liquid to a levelhigh enough to completely submerge the the cup by the action of thedisturbance set'up in the liquid when the cup is rotated at relativelyhigh rates of speed and means for rotating said cup.

4. Tube spinning apparatus as set forth in claim 3 in which the cup isprovided, below the mouth portion thereof, with flow openings placingthe cavity of the cup in communication with the interior of the casing.

ROBERT REDVERS THOMPSON.

